The non-chromosomal genes URE3, PSI and PIN of Saccharomyces cerevisiae are infectious forms (prions) of the Ure2p, Sup35p and Rnq1p, respectively, while Het-s of Podospora anserina is a prion of the HETs protein (1-3, reviewed in 4). The basis of each of these prions is a self-propagating amyloid of the respective protein (5-8), as the mammalian infectious protein PrPSc is believed to be an amyloid of the cellular PrP protein. [unreadable] Amyloid is a filamentous protein form characterized by high beta-sheet content, unique staining properties with the dye Congo Red, and partial protease resistance. Because these filaments are large and insoluble, they are not suitable material for either X-ray crystallography or solution NMR structural studies. However, sophisticated solid-state NMR methods are beginning to allow study of materials such as these (reviewed in ref. 9). We have undertaken solid-state NMR studies of each of the yeast and fungal prion amyloids in collaboration with Dr. Robert Tycko of LCP, NIDDK. The first of these studies shows that infectious amyloid of the Sup35 protein, the basis of the PSI prion, is a parallel in-register beta-sheet structure (10).[unreadable] Sup35p is a subunit of the translation termination factor that recognizes termination codons on mRNA, cleaves the completed peptide from the last tRNA and releases it from the ribosomes. The Sup35 protein consists of an N-terminal prion domain called N (residues 1-123), a middle (M) highly charged domain (residues 124 - 253) and a C-terminal domain (called C) that is sufficient for translation termination. Sup35NM is sufficient for infectivity and was used in our studies. We prepared recombinant Sup35NM labeled with tyrosine-1-13C, or leucine-1-13C, or phenylalanine-1-13C, and made amyloid filaments from each. One dimensional 13C NMR spectra of these filaments showed that the carbonyl carbon nuclei were shifted to lower resonant frequencies indicative of each being in beta sheet structure.[unreadable] Beta sheets include antiparallel sheets (the most common in soluble enzymes), parallel beta sheets, and beta helices. Parallel beta sheets may be either in-register or out of register. An in-register beta sheet has identical residues aligned along the filament. For example, Val31 of the first molecule is 4.7 angstroms (the distance between beta strands in a beta sheet) from Val31 of the second molecule and so on down the fiber. [unreadable] We used a constant-time finite-pulse radio frequency driven recoupling method (11) to measure the distance from each labeled carbonyl carbon to the next nearest labeled nucleus. In this method, the rate of decay of the NMR signal is inversely proportional to the cube of the distance between the labeled nuclei. We found that for tyrosine-1-13C and for leucine-1-13C, the decay indicated a distance of about 5 angstroms, demonstrating a parallel beta sheet structure, either in register or out of register by at most one residue (10). Diluting the fully labeled molecules with four parts of unlabeled molecules resulted in substantial decrease in the rate of signal decay, indicating the the interaction being observed was intermolecular, not intramolecular, and again supporting the parallel in-register structure. Ether-precipitated Sup35NM labeled with Leu-1-13C showed a slow decay rate indicating that the results observed were due to the specific amyloid structure, not simply due to aggregation.[unreadable] To confirm that the parallel sheet was indeed in-register, we made Sup35NM labeled with alanine-3-13C, that is, methyl labeled instead of carbonyl carbon labeled as in the other experiments. Because amino acid R groups point in alternate directions with succeeding residues on a beta strand, a parallel beta sheet out of register by a single residue would give a very slow decay rate. In fact the results indicated an in-register structure (10). We also found, unexpectedly, that, in addition to the prion domain N, the M domain has considerable parallel in-register beta sheet structure (10).[unreadable] This is the first structure determined for any prion amyloid. Further work will be needed to determine the details of this structure, but specifying a parallel in-register structure constrains the possibilities very tightly. We have carried out similar studies on infectious amyloid of the Ure2 prion domain (residues 1-89) with similar results (Baxa et al., in press). Work is now in progress on amyloid of the Rnq1 protein, the basis of the PIN prion of yeast, and the prion domain of HETs.[unreadable] Implications:[unreadable] In the parallel in-register beta sheet structure, each residue of the prion domain contacts the same residue in the molecule that was laid down before it and the same residue in the next molecule to join the filament. This provides the possibility of passing on structural variations - such as turns of the sheet or irregularities in the structure - from parent molecules (already in the filament) to daughter molecules newly being laid down. This is the essential "templating" mechanism that must be present for these proteins to act as genes. This can explain the prion "variants", cases where the identical amino acid sequence produces different phenotypes or prion stabilities, doubtless due to differing details of the amyloid structure.[unreadable] This structure also indicates that the sequence of events is not, as often suggested, a primary conformational change of the prion protein, followed by aggregation. It seems evident that the conformational change is coincident with and a consequence of the new molecule joining the filament, and the details of the conformational change are directed by the structure of the end of the filament.[unreadable] [unreadable] 1. Wickner, R. B. URE3 as an altered URE2 protein: evidence for a prion analog in S. cerevisiae. Science 264, 566 - 569 (1994).[unreadable] 2. Coustou, V., Deleu, C., Saupe, S., and Begueret, J. (1997). The protein product of the het-s heterokaryon incompatibility gene of the fungus Podospora anserina behaves as a prion analog. Proc. Natl. Acad. Sci. U. S. A. 94, 9773 - 9778.[unreadable] 3. Derkatch, I.L., Bradley, M.E., Hong, J.Y., and Liebman, S.W. (2001). Prions affect the appearance of other prions: the story of PIN. Cell 106, 171 - 182.[unreadable] 4. Wickner, R.B., Edskes, H.K., Ross, E.D., Pierce, M.M., Baxa, U., Brachmann, A., and Shewmaker, F. (2004). Prion Genetics: New Rules for a New Kind of Gene. Ann. Rev. Genetics 38, 681-707.[unreadable] 5. Maddelein, M.L., Dos Reis, S., Duvezin-Caubet, S., Coulary-Salin, B., and Saupe, S.J. (2002). Amyloid aggregates of the HET-s prion protein are infectious. Proc Natl Acad Sci U S A 99, 7402-7407.[unreadable] 6. King, C.Y., and Diaz-Avalos, R. (2004). Protein-only transmission of three yeast prion strains. Nature 428, 319 - 323.[unreadable] 7. Tanaka, M., Chien, P., Naber, N., Cooke, R., and Weissman, J.S. (2004). Conformational variations in an infectious protein determine prion strain differences. Nature 428, 323 - 328.[unreadable] 8. Brachmann, A., Baxa, U., and Wickner, R.B. (2005). Prion generation in vitro: amyloid of Ure2p is infectious. Embo J 24, 3082 - 3092.[unreadable] 9. Tycko, R. (2006). Molecular structure of amyloid fibrils: insights from solid-state NMR. Q. Rev. Biophys. 1, 1-55.[unreadable] 10. Shewmaker, F., Wickner, R.B., and Tycko, R. (2006). Amyloid of the prion domain of Sup35p has an in-register parallel &#61538;-sheet structure. Proc. Natl. Acad. Sci. U. S. A. 103, 19754 - 19759.[unreadable] 11. Balbach, J.J., Petkova, A.T., Oyler, N.A., Antzutkin, O.N., Gordon, D.J., Meredith, S.C., and Tycko, R. (2002). Supramolecular structure in full-length Alzheimer's beta-amyloid fibrils: Evidence for a parallel beta-sheet organization from solid-state nuclear magnetic resonance. Biophys. J. 83, 1205-1216.